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CROP QUALITY & UTILIZATION

Selection and Evaluation of Smooth Bromegrass Clones with Divergent Lignin or Etherified Ferulic Acid Concentration

^a Dep. of Agronomy, 1575 Linden Dr., Univ. of Wisconsin, Madison, WI 53706-1597 USA
^b USDA-ARS Plant Science Res. Unit and US Dairy Forage Res. Ctr. Cluster, Dep. of Agronomy and Plant Genetics, 411 Borlaug Hall, 1991 Upper Buford Cir., Univ. of Minnesota, St. Paul, MN 55108 USA

mdcasler{at}facstaff.wisc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Lignin and etherified ferulic acid (EthFA) are cell wall constituents believed to have important negative impacts on digestibility of forage cell walls. Our objective was to identify genotypes of smooth bromegrass (Bromus inermis Leyss) with unconfounded divergence in lignin and EthFA, such that their independent effects could be determined on in vitro fiber digestibility (IVFD). Eight clones were selected from each of four populations (`Alpha', WB19e, `Lincoln', and WB88S). Selection was successful for EthFA in all populations except WB88S, creating repeatable divergence of 11.2 to 12.5%. Selection was unsuccessful for lignin concentration per se, most likely because of large genotype x environment interactions. Nevertheless, the resulting clones showed significant variation for both EthFA and lignin concentrations and these two variables were nearly independent among the selected clones. Both lignin and EthFA had significant negative effects on 96-h IVFD, regardless of the statistical estimation method used. Across estimation methods, there was not a clear difference in the magnitude of the lignin and EthFA effects on IVFD. Both the total amount of lignin in the cell wall and the amount of ferulic acid cross-linking lignin to polysaccharides via ether bonds appear to be under genetic control, and both components play a role in regulating the genetic potential for rumen degradation of cell walls in smooth bromegrass. These clones are a potential tool for conducting animal feeding trials using forages with relatively unconfounded differences in a single cell wall characteristic.

Abbreviations: EthFA, Etherified ferulic acid • IVFD, in vitro fiber digestibility • NDF, neutral detergent fiber

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
LIGNIFICATION

is one of the major causes of reduced digestibility in forage crops. While this point is scarcely debatable, the precise mechanism by which lignification reduces digestibility is unclear. Lignin itself may form a physical barrier to prevent degradation of polysaccharides by rumen enzymes (Cowling, 1975; Van Soest, 1973). Covalent cross-linkages between lignin and arabinose units of xylan chains by ferulic acid bridges may limit enzymatic degradation by positioning the lignin in proximity to cell-wall polysaccharides (Jung and Deetz, 1993).

In smooth bromegrass, genetic progress from selection for high in vitro digestibility has been largely due to decreases in lignin concentration (Casler and Vogel, 1999). Among high-digestibility selections, variation in digestibility was due largely to variation in lignin concentration (Jung and Casler, 1990, 1991). However, among low-digestibility selections, the influence of lignin concentration was dramatically reduced and individual cell-wall hydroxycinnamic acids became more important. In particular, esterified ferulic acid concentration showed a strong and negative linear relationship with lignin concentration (Jung and Casler, 1990), but appeared to be more associated with fiber digestibility than was lignin concentration (Jung and Casler, 1991). In vitro fiber digestibility was negatively infuenced by lignin concentration, but positively influenced by esterified ferulic acid concentration (Jung and Casler, 1990, 1991). In switchgrass (Panicum virgatum L.), high-digestibility genotypes had higher ratios of esterified ferulic to p-coumaric acid than low-digestibility genotypes (Gabrielson et al., 1990), a pattern similar to that observed between normal and high-digestibility brown-midrib (bm3) maize (Barrière and Argillier, 1993).

These results suggest that ferulate cross-linkages between arabinoxylans and lignin may be more important in regulating genetic variation for digestibility than the concentration per se of these constituents (Jung and Deetz, 1993). Ferulic acid is esterified to arabinose subunits of arabinoxylan chains while p-coumaric acid is primarily esterified to lignin during plant development (Jung, 1989). These ferulate-polysaccharide esters act as nucleation sites for lignification and may regulate the degree of lignin-polysaccharide cross-linking that occurs as plants develop (Ralph et al., 1995). Because the concentrations of lignin (Jung and Casler, 1990) and polysaccharide (Godshalk et al., 1988) monomers appear to be under genetic control in forage crops, it is theoretically possible to create genotypes with an altered rate of lignin-polysaccharide cross-linkage formation during development.

As plants mature, lignin synthesis increases lignin concentration (Casler, 1986) and esterified ferulates become etherified to lignin, forming cross-linkages between lignin and cell-wall polysaccharides (Iiyama et al., 1990). Both of these processes are thought to limit the digestion of cell-wall polysaccharides by rumen cellulolytic microorganisms (Jung and Deetz, 1993). In studying the genetics of cell-wall development and digestion, hundreds of plant samples must be taken at a given maturity stage, limiting our ability to study ontogeny of cell-wall development. Such sample sets provide a picture of ontogenic development at the chosen maturity stage, which occurs on the same date for most smooth bromegrass genotypes (Casler and Vogel, 1999). Environmental replication (multiple harvests and replicates) broadens the inferences of such a study, but does not alter its basic structure. The challenge in identifying single genetic factors that influence digestibility is to limit the confounding factors that vary at that single point in time.

There are three biochemical–physiological processes that are ontogenically confounded, each of which greatly influences digestibility (Buxton and Casler, 1993): (i) advancement of reproductive maturity, (ii) changes in the relative proportions of cell types toward a greater frequency of structural support tissues, and (iii) thickening and strengthening of cell walls per se. In smooth bromegrass, there is no genetic variation for timing of reproductive maturity, so any genetic variation in digestibility or cell-wall development is independent of this trait. Although there is considerable variation in smooth bromegrass stem and leaf blade anatomy, little of this variation is related to digestibility (Ehlke and Casler, 1985). These authors' work clearly shows that variation in chemical traits is considerably more important than variation in anatomical traits (relative proportion of rapidly vs. slowly digested cell types) in regulating digestibility. Thus, it is likely that genotypic variation for digestibility at any individual sampling date in smooth bromegrass is largely due to genotypic variation in the rate of cell-wall ontogenic development preceding the sampling date.

Our objective was to identify genotypes of smooth bromegrass with unconfounded divergence in lignin and EthFA, such that their independent effects can be determined on in vitro fiber digestibility (IVFD). Our approach was to break the apparent genetic correlation between lignin and EthFA concentration by practicing divergent selection for four phenotypes: high lignin-high EthFA, high lignin-low EthFA, low lignin-high EthFA, and low lignin-low EthFA. We based all selection and evaluation on vegetative plant samples consisting primarily of leaf blades to reflect the predominant plant parts available in well-managed grazing systems.

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Stage 1 Selection
Selection was applied to four smooth bromegrass populations: the cultivars Alpha and Lincoln, and the synthetic populations WB19e and WB88S (Falkner and Casler, 1998). Three hundred 70-d-old seedlings of each population were transplanted to the field in May 1992. Plants were arranged into 10 blocks of 30 plants each with a spacing of 0.9 m between all adjacent plants. The experiment was located at Arlington, WI on a Plano silt loam (fine-silty, mixed, mesic, Typic Argiudoll). Weeds were controlled by pre-emergence herbicide applications (Falkner and Casler, 1998). Plants were fertilized with 112 kg N ha^-1 in late June.

A leaf tissue sample was clipped from each plant at a vegetative growth stage in mid-August, with a stubble height of 4 cm. Tissue samples were placed in paper bags and dried at 60°C. Dried samples were ground through a 1-mm screen of a Wiley-type mill and re-ground through a 1-mm screen of a cyclone mill. Two independent subsets of each sample were scanned on a near-infrared reflectance spectrometer (NIRS, Pacific Scientific Model 51A, Silver Spring, MD). Two random plants from each block of each population comprised a stratified random subset of 80 plants that was subjected to wet-laboratory analysis. Klason lignin concentration was measured as the ash-free residue remaining after cell-wall polysaccharide hydrolysis (Theander et al., 1995). Esterified ferulic acid concentration in the cell wall was determined by 2 M NaOH extraction and HPLC analysis (Jung and Shalita-Jones, 1990). Concentration of etherified ferulic acid (EthFA) was computed as the difference between total ferulic acid, obtained by 4 M NaOH extraction at 170°C for 2 h, and the esterified fraction (Iiyama et al., 1990). Neutral detergent fiber (NDF) concentration was determined by the procedure of Van Soest et al. (1991), omitting the sodium sulfite and -amylase steps. Etherified ferulic acid and NDF were determined on duplicate samples, while Klason lignin was determined on quadruplicate samples. Means over laboratory replicates were used to calibrate the NIRS for prediction of the entire set of 2400 scanned samples (four populations x 300 plants x two scanned subsets). Means over the two scanned subsets of each sample were computed prior to selection.

Some field and laboratory variability was removed from the estimates of plant phenotypes by computing t-scores to adjust for differences among block means (Casler, 1992):

where t_ij is the t-score for lignin or ferulic acid of the ijth plant, X_ij is the raw datum for the ijth plant, M_j is the mean of the jth block, and s_j is the standard deviation of the jth block. Plants were selected on the basis of the most extreme values of t_ij for Klason lignin and EthFA to fall within the four groups: high lignin-high EthFA (HL-hf), high lignin-low EthFA (HL-lf), low lignin-high EthFA (LL-hf), and low lignin-low EthFA (LL-lf).

Stage 2 Selection
Eighty plants were selected from Stage 1, five per group within each population. Plants were fertilized again in late April 1993 with 56 kg N ha^-1. These plants were resampled in mid-May 1993 by the same procedure as described for the August 1992 sampling. Each plant was analyzed using the same wet-laboratory procedures applied to the 1992 samples. Based on the mean over the two sampling dates, 32 clones were selected, two from each group (HL-hf, HL-lf, LL-hf, and LL-lf) within each population.

Stage 2 Evaluation and Stage 3 Selection
These 32 clones were clonally replicated and transplanted to a randomized complete block design with two replicates at Arlington in May 1994. Each propagule consisted of a 100-cm² cylinder of sod, 10 cm deep. Plants were spaced 0.9 m apart. Plants were clipped without harvesting in July and September 1994 and in June 1995 and 1996. Fertilizer was applied following clipping in June and August 1995 and 1996 at a rate of 56 kg N ha^-1 for each application. Leaf tissue samples were harvested as described above in early August and late September 1995 and 1996. All samples were dried, ground, and analyzed according to wet-laboratory procedures as previously described. Etherified and esterified p-coumaric acid (PCA) concentrations were determined as described for ferulic acid. In vitro digestibility of the NDF was determined for 24- and 96-h fermentations according to the procedure of Jung et al. (1992). Fermentation times of 24 and 96 h were used to provide estimates of rapidly and potentially digestible NDF fractions, respectively.

Data from the original nursery were subjected to a one-way analysis of variance to estimate the efficiency of the grid selection system (Casler, 1992). Data from the randomized complete block experiment were subjected to a split-plot-in-time analysis of variance combined over harvests and years (Steel et al., 1996). Phenotypic correlation coefficients were used to study the relationships among variables and years within variables. Homogeneity of phenotypic correlation coefficients was tested as described by Steel et al. (1996).

The effect of Klason lignin and EthFA on in vitro NDF digestibility (IVFD) was studied by three statistical methods. First, a priori contrasts within the analyses of variance for IVFD were formed to test the effect of divergent selection for lignin or EthFA. Second, standardized partial least squares (Draper and Smith, 1981) was used to estimate the standardized and independent effects of all cell wall constituents on IVFD. Third, individual pairs of clones divergent in lignin concentration (P < 0.01) and similar in EthFA (P > 0.50), or vice versa, were used to create a posteriori contrasts which were applied to the IVFD data within each population.

	Results and discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Stage 1 Selection
There was considerable phenotypic variation for lignin, EthFA, and NDF within all four populations, as indicated by the range and standard deviation (Table 1) . Phenotypic coefficients of variation ranged from 5.3 to 6.0% for lignin, 6.2 to 6.6% for EthFA, and 3.9 to 4.6% for NDF. Blocking removed environmental variation for all three variables with the following ranking: lignin > EthFA > NDF. There were no consistent differences among populations in phenotypic variability (PSD) or the effectiveness of blocking (R²).

The replicated experiment showed further evidence of GxE interaction, both internally, among harvest dates, and externally, with the original selection nursery (Table 4) . For lignin, GxE effects were greatest between the two plantings (1992-1993 vs. 1995-1996). Although the August 1995 date was not correlated with either of the two 1996 dates, the remainder of the correlations suggested minor internal GxE interactions. For EthFA, all environmental factors contributed to GxE interaction, including harvest date, year, and field site. For NDF, the most important GxE interactions were between specific site-harvest-year combinations (May 1993 vs. 1995-1996 and October 1995 vs. all 1996). Despite these interactions, means over all 1995-1996 harvest dates provided the best estimate of clonal performance and were used for all additional data analyses. Data from 1992-1993 were not used further because the clones were unreplicated in the original nursery and their use would inflate any estimates of true genetic differences among clones.

In contrast, divergent selection for EthFA concentration was highly successful (Table 5). Clones originally classified as "high" or "low" in EthFA concentration generally retained their classification in the replicated experiment, with mean differences significant for all but WB88S. High-EthFA clones averaged 3.7 to 12.5% higher in EthFA than low-EthFA clones. Although these responses do not allow conclusions about the heritability of EthFA concentration, they suggest some optimism for the prospects of continued selection for either high or low EthFA concentration.

Divergent selection for EthFA concentration showed some correlated responses, more likely due to the restricted number of genotypes than to the presence of true genetic correlations (i.e., sampling variation). Divergent EthFA clones coincidentally differed in Klason lignin concentration for Alpha and WB19e and for NDF in all four populations (Table 5). However, the fact that half of these effects were positive and half negative suggested more of a random process than a biologically meaningful relationship. This result and the correlation coefficients in Table 2 suggested that, had we the luxury of a larger population size, these correlated responses would likely have been smaller or nonexistent.

Phenotypic correlation coefficients among the cell-wall components measured in this study were generally homogeneous across populations, so were computed for all 32 clones (Table 6) . Clones with more total fiber tended to have higher total amounts of all four hydroxycinnamic acid components: both etherified and esterified ferulic acid (FA) and PCA. However, high-NDF clones only showed a slight trend toward higher concentration of hydroxycinnamic acids within the NDF fraction. The etherified forms of FA and PCA were highly correlated within the NDF fraction, suggesting some degree of generality in the etherification process. The esterified and etherified forms of PCA were also highly correlated , indicating possible ontogenic coincidence of PCA esterification and etherification. These two processes were not associated for FA . The difference in correlation between ester- and ether-linked forms of PCA and FA was expected. Ferulic acid esters are deposited primarily (possibly exclusively) during primary cell-wall growth, whereas FA ethers and both forms of PCA are deposited primarily during secondary cell-wall deposition (Jung and Deetz, 1993; Morrison et al., 1998).

Standardized partial least squares regression allows estimation of the direct (independent) effects of each cell-wall or fiber component on IVFD. Because of limited degrees of freedom, the full model of all cell-wall components could only be tested on all 32 clones. Because lignin and EthFA showed the only evidence for significance in the full model, all other components were removed and the model was recomputed separately for each population (Table 8) . As with the a priori contrasts, there was little evidence of a relationship of lignin or etherified ferulic acid with 24-h IVFD. Conversely, direct effects of lignin on 96-h IVFD were significant in three of four populations, excluding WB88S, ranging from -1.01 to -0.50. Direct effects of etherified ferulic acid on 96-h IVFD were always lower than those for lignin and only two of four values were significant. On the basis of the standardized partial regression coefficients for Alpha, WB19e, and Lincoln, lignin was 1.1 to 1.9 times more important than EthFA in controlling 96-h IVFD. Both cell-wall components had consistently negative effects on IVFD. The value of these regression coefficients can be seen in their consistency relative to the correlation coefficients, which are not adjusted for covariation with other variables.

Stage 2 Evaluation and Stage 3 Selection
The a priori contrasts are useful in describing selection responses per se, but they suffer somewhat from a lack of independence of the two selection criteria. A posteriori comparisons allow the development of specific comparisons based on single-variable divergence without confounding effects. In the four populations, five two-clone comparisons could be formed with significant (P < 0.01) EthFA divergence and non-significant (P > 0.50) lignin divergence (Table 9) . Each of these comparisons showed an opposite change in 96-h IVFD, with the four significant effects ranging from -47 to -22 g kg^-1 of 96-h IVFD for each 1 g kg^-1 of EthFA divergence.

View larger version (30K): [in this window] [in a new window]   Fig. 1 Scatterplots of etherified ferulic acid vs. Klason lignin concentration for clone means of four smooth bromegrass populations averaged over four harvests in 1995 and 1996. Doubleheaded arrows represent LSD values for clone means within populations (P < 0.01). Closed symbols represent the most desirable clones within each population for testing the effect of different concentrations of lignin at constant EthFA or different concentrations of EthFA at constant lignin. Numbers refer to pairwise comparisons made in Table 9

These a posteriori comparisons provided a basis for conducting a third stage of selection among these clones. We failed at our original selection goal of identifying extreme genotypes (HL-hf, HL-lf, LL-hf, and LL-lf) along the two diagonal axes of the lignin-etherified ferulic acid bivariate distribution (Fig. 1) . Instead, we were successful at identifying groups of clones for which the apparent genetic relationship between lignin and etherified ferulic acid concentrations (Jung and Casler, 1990) was broken. Clones with extreme divergence along vertical (EthFA) or horizontal (lignin) axes were readily identified. Furthermore, many of these differences were not confounded between the two selection criteria and were associated with 96-h IVFD (Table 9). Because of their divergence and low LSD values, Alpha and WB19e provided the most promising populations from which to select a group of four clones that could be vegetatively increased for use in animal feeding trials. Of these two, WB19e provided the largest differences among clones and the greatest level of independence between the two selection criteria.

	Conclusions

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Selection for divergent EthFA was successful in three of four populations, creating clones with divergent EthFA at relatively constant lignin concentration. Although we obtained repeatable differences among clones for lignin concentration and lignin concentration was independent of EthFA concentration, future selection for Klason lignin concentration will require a more rigorous protocol. The GxE interactions for Klason lignin suggested that lack of proper environmental replication during selection can lead to divergence that is environment specific and not broadly repeatable. Broad repeatability of differences is critical to such a selection program, because these clones must eventually be fed to animals to follow this research to its logical conclusion.

Despite our problems in identifying repeatable divergence for lignin, these clones represent a potentially valuable tool for studying ruminant nutrition. Relationships of both lignin and EthFA with IVFD suggested that both the total amount of lignin and the amount of ferulic acid cross-linking lignin with cell-wall polysaccharides are important determinants of digestibility. The genetic independence that we achieved by selection provided assurance that these effects are reasonably independent of each other. The amount of ferulic acid etherified to lignin is not dependent on lignin concentration per se, nor the converse. In vitro fiber digestibility of a smooth bromegrass plant can be reduced by (i) additional ferulate cross-linking, (ii) additional biosynthesis of lignin per se, or (iii) a combination of both processes. Because these clones can be vegetatively propagated with relative ease, it is possible to produce forage of each clone in sufficient quantity for animal feeding trials. Such a trial would allow a specific test of the independent effects of lignin or ferulate-mediated cross-linking on animal performance. The clones from WB19e appear to offer the greatest potential for this purpose.

Received for publication December 29, 1998.

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TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 

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